The specific embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.
The preparation of embodiment 1, oligomannuronic acid
The present invention adopts the acid degradation method to prepare and separate the polymannuronate that obtains high molecular from the fucoidin Algin, further obtains oligomannuronic acid among the present invention through acid degradation or enzymatic degradation again.
The Algin water is mixed with the glue of concentration 2%, in the 0.5mol/L hydrochloric acid solution in 100 ℃ of hydrolysis 10 hours, behind the non-shock chilling, abandoning supernatant.To be precipitated and dissolved in 2%Na
2CO
3In the aqueous solution, regulate pH value to 2.85, the centrifugal precipitation of removing with dilute hydrochloric acid.Then add hydrochloric acid acid concentration to the system in the supernatant and reach 0.3mol/L, stir degraded 5 hours at 100 ℃.Then separate taking precipitate, will be precipitated and dissolved in 2%Na
2CO
3In the aqueous solution, with dilute hydrochloric acid adjust pH to 2.85, recentrifuge is removed precipitation.Then supernatant is added the long-pending ethanol of tetraploid and precipitate, it is formula I that precipitation is drying to obtain oligomannuronic acid.Can further this oligomannuronic acid be made 5% solution, add the reaction of alginate lyase liquid after 30 minutes, heat inactivation in 28 ℃.Then add the long-pending ethanol of tetraploid and precipitate and the while desalination, the oligomannuronic acid that precipitation is drying to obtain enzymolysis is formula II.
The oligomannuronic acid that the present invention makes or the molecular skeleton of its pharmaceutical salts are β-D-(1 → 4) the mannopyranose aldehydic acid that connects, weight average molecular weight is<10kDa, the non reducing end saccharide residue can contain 4,5-position unsaturated double-bond, the concrete structure formula is as follows, R=H or Li in the formula, Na, K, Ca, Mg pharmaceutical salts.
Oligomannuronic acid high-efficient liquid phase analysis spectrogram as shown in Figure 1, prepared oligomannuronic acid weight average molecular weight is 5401, breadth coefficient (D) is 1.11.
The oligomannuronic acid infrared spectrum is as shown in Figure 2, and listed absworption peak ownership among the figure is as follows:
Oligomannuronic acid carbon-13 nmr spectra figure as shown in Figure 3, the 177.15ppm place is C6 position carboxyl carbon signal among the figure, the 101.83ppm place is the C1 signal, 79.67ppm locate the signal into C4,77.76ppm locate the signal into C5, the 73.24ppm place is the signal of C3, the 71.89ppm place is the C2 signal.
Embodiment 2, oligomannuronic acid reduce the effect of disease to the caused by cyclophosphamide murine interleukin
According to internationally recognized method, estimated it the murine interleukin of caused by cyclophosphamide reduced the effect of disease, and on cellular level preliminary study its mechanism of action.
Mouse peritoneal injection cyclophosphamide causes the leukopenia model, observe Oligomeric manna sugar aldehyde to the impact of quantity of leucocyte in the mouse peripheral blood, and measure mouse bone marrow cells nucleated cell quantity, having studied simultaneously the medicine vivo medicine-feeding is the impact of hemopoietic progenitor cell colony on mice grain-huge biting.
Concrete experimental technique is as follows: 60 of Kunming mouses are divided into 6 groups at random by body weight, Normal group, model control group, lithium carbonate positive controls, three dosage groups of oligomannuronic acid.Each treated animal gavage (ig) administration, normal group and model group give distilled water.Administration 9d, except Normal group, each organizes equal lumbar injection (ip) cyclophosphamide 50mg/kg, continuous 3d, last to cyclophosphamide after the 3rd day, carry out following experiment:
1) each treated animal tail point is got blood 20ul, adds the 1ml blood dilution liquid, measures hemogram on blood count, the results are shown in Figure 4.Among Fig. 4, except normal group, all the other each groups are all injected the cyclophosphamide modeling.Normal group and model group give water, and positive group gavage gives lithium carbonate, and dosage is 180mg/g; The dosage of basic, normal, high three the dosage groups of oligomannuronic acid is respectively 180mg/kg, 360mg/g and 540mg/kg.Vertical coordinate represents leukocytic quantity in the mouse peripheral blood among the figure.Compare ##, P<0.01 with Normal group.Compare *, P<0.05 with model group; *, P<0.01.
As seen from Figure 4, ip in mice cyclophosphamide 50mg/kg, 3d can make the mouse peripheral blood leukocyte obviously descend continuously, compares with Normal group, and utmost point significant difference is arranged.Mice took oligomannuronic acid 14 days continuously, and three dosage groups can both leukocyte increasing reduce the quantity of leucocyte of disease mice, with model group significant difference and utmost point significant difference, P<0.05 and P<0.01 are arranged relatively.It can also be seen that from Fig. 4 the effect of oligomannuronic acid leukocyte increasing is better than the positive drug lithium carbonate.
2) after mice is put to death, put in 75% ethanol and sterilize, separate a side femur under the aseptic condition, cut off the femur two ends, draw the RPMI-1640 culture fluid with the 1ml asepsis injector, medullary cell is gone out, blow and beat into unicellularly with No. 4 syringe needles, at microscopically counting bone marrow nucleated cell number, the results are shown in Figure 5.Among Fig. 5, grouping and the same Fig. 4 of administration situation.Vertical coordinate represents mouse bone marrow cells nucleated cell quantity.Compare ##, P<0.01 with Normal group.Compare * *, P<0.01 with model group.
As can be seen from Figure 5, leukocytic variation tendency is consistent in the variation tendency of each treated animal bone marrow nucleated cell number and the peripheral blood.The bone marrow nucleated cell number of intact animal's a side femur reaches 10.69 * 10
6Individual, and behind the injection cyclophosphamide, bone marrow nucleated cell quantity obviously reduces in the model group animal femur, nucleated marrow cell population only is 4.49 * 10 in the side femur
6Individual, compare with normal group, utmost point significant difference is arranged.Three dosage groups of oligomannuronic acid can obviously increase mouse bone marrow cells nucleated cell quantity, compare with model group, utmost point significant difference are arranged, P<0.01.
3) utilize external agar culture method to observe the oligomannuronic acid vivo medicine-feeding to the impact of mice CFU-GM.Kunming mouse is taken off cervical vertebra puts to death, put in 75% the ethanol behind the sterilization 5min, separate femur under the aseptic condition, cut off the femur two ends, draw the RPMI-1640 culture fluid with the 1ml asepsis injector, go out medullary cell, blow and beat into unicellular with No. 4 syringe needles, 1000 abandon culture fluid after leaving heart 5min, add the RPMI-1640 culture fluid and adjust cell to suitable concn.The results are shown in Figure 6.Among Fig. 6, vertical coordinate represents CFU-GM quantity.Compare ##, P<0.01 with Normal group.Compare * *, P<0.01 with model group.
As can be seen from Figure 6, the cyclophosphamide model group causes the hemopoietic system of mice to suffer damage, and hemopoietic progenitor cell quantity obviously reduces in its bone marrow, and the colony of generation and normal group compare, and quantity is few, and contained cell quantity also lacks than normal group in the single colony.Can obviously the raise quantity of CFU-GM of three dosage groups of oligomannuronic acid, with model group relatively, utmost point significant difference is arranged.And the effect of oligomannuronic acid low dose group is suitable with positive drug carbonic acid Role of lithium, and the effect of middle high dose group is better than lithium carbonate.
Embodiment 3, oligomannuronic acid treated in vitro are on the impact of CFU-GM
Utilize external agar culture method, studied the oligomannuronic acid treated in vitro, on the impact of normal bone marrow cells in mice and cyclophosphamide inhibition bone marrow cells in mice CFU-GM.
1) effect that normal bone marrow cells in mice CFU-GM is formed: get normal mouse, the conventional medullary cell that separates is got the RPMI-1640 culture fluid medullary cell is adjusted to concentration 1 * 10
6Ml
-1Carrying out colony cultivates.Experimental group adds respectively the medicine of variable concentrations, makes its final concentration be respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, and matched group adds the RPMI-1640 culture fluid with volume.Above-mentioned cultivating system is added in 24 orifice plates, every hole 0.5ml, each concentration is established 3 multiple holes, puts 37 ℃, 5%CO
2Cultivate in the incubator.After 5 days, carry out colony count under inverted microscope, all colonies that contains 50 above cells are a CFU-GM colony.The results are shown in Figure 7a and Fig. 7 b.Among Fig. 7 a, blank group is the matched group of not dosing; The dosage of five administration groups of oligomannuronic acid is respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, and vertical coordinate represents the quantity of CFU-GM colony.Compare * *, P<0.01 with the blank group; *, P<0.05.
Can find out from Fig. 7 a and Fig. 7 b, utilize the agar extracorporeal culture-ing, oligomannuronic acid can obviously promote the formation of CFU-GM colony, compares with the blank group, and significant difference is arranged.And arrive in the 25ug/ml scope 3.125, along with the increasing of drug level, effect also strengthens gradually, and effect reaches maximum during 25ug/ml.
2) cyclophosphamide is suppressed the effect that bone marrow cells in mice CFU-GM forms: get normal mouse, the conventional medullary cell that separates is got the RPMI-1640 culture fluid medullary cell is adjusted to concentration 1 * 10
6/ ml carries out colony and cultivates.Except Normal group, it is the cyclophosphamide of 100uM that other each groups add final concentration in the cultivating system.Experimental group adds respectively the medicine of variable concentrations, makes its final concentration be respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, and normal group and cyclophosphamide model group add the RPMI-1640 culture fluid with volume.Cultivate after 5 days, under inverted microscope, carry out colony count.The results are shown in Figure 8.Among Fig. 8, normal group is the matched group of not dosing; The CY group is not dosing, adds the model control group of cyclophosphamide (CY); The dosage of five administration groups of oligomannuronic acid is respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, adds simultaneously CY.Vertical coordinate represents the quantity of CFU-GM colony among Fig. 8.Compare ##, P<0.01 with Normal group.Compare * *, P<0.01 with the blank group; *, P<0.05.
Each hemopoietic progenitor cell colony that the agar extracorporeal culture-ing forms derives from a cell.Granulocyte is that proliferation of hematopoietic progenitors differentiates by grain-huge biting, so the formation of In vitro culture observation CFU-GM, can reflect the size of Leukocytopoiesis ability.CFU-GM is more, illustrates that body has more hemopoietic progenitor cell, and also just having more cell to come Development And Differentiation is leukocyte.
As can be seen from Figure 8, the cyclophosphamide of 100uM can obviously suppress the formation of mouse bone marrow cells CFU-GM, compares with normal group, and significant difference is arranged.Can obviously the raise quantity of CFU-GM of oligomannuronic acid, with model group relatively, utmost point significant difference is arranged.And in the 25ug/ml scope, obvious dose-effect relationship is arranged at 3.125ug/ml, namely along with the increase of drug level, it promotes the effect of CFU-GM to strengthen gradually, reaches maximum effect during to 25ug/ml.
Embodiment 4, oligomannuronic acid are on the impact of Marrow Stromal Cells in Proliferation
Utilize mtt assay, studied the impact of oligomannuronic acid on mouse bone marrow propagation.
Get 1 of mice, the conventional medullary cell that separates is adjusted cell concentration to 2 * 10 with the RPMI-1640 culture fluid of 20%FBS
6/ ml.Get 96 orifice plates, every hole adds 180ul cell suspension, adds respectively the oligomannuronic acid of 20ul variable concentrations again, make final concentration be respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, the blank group adds isopyknic culture fluid.Cell is placed 37 ℃, 5%CO
2Cultivate in the incubator after 7 days, mtt assay is measured the propagation of marrow stromal cell.The results are shown in Figure 9.Among Fig. 9, blank group is the matched group of not dosing; The dosage of oligomannuronic acid administration group is respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml.Among Fig. 9, vertical coordinate represents the absorbance at 570nm place.Compare * *, P<0.01 with the blank group; *, P<0.05.
As can be seen from Figure 9, Oligomeric manna sugar aldehyde has obvious promotion proliferation function to mouse bone marrow, and except 3.125ug/ml concentration, other four concentration groups and blank group more all have notable difference.
Embodiment 5, oligomannuronic acid are to the effect of marrow stromal cell secrete GM-CSF
Utilize the ELISA method to measure the content of stromal cell secrete GM-CSF.
Get the RPMI-1640 culture fluid that contains 20% hyclone, it is 2 * 10 that medullary cell is adjusted to concentration
6/ ml.Get 6 orifice plates, every hole adds bone marrow cell suspension 1.8ml, the oligomannuronic acid that adds again the 0.2ml variable concentrations, make its final concentration be respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, matched group adds the culture fluid of equivalent, and each group is established respectively 3 multiple holes.Cell is placed 37 ℃, 5%CO
2After cultivating 8d in the incubator, draw culture supernatant, behind 4 ℃ of low-temperature centrifugations, get supernatant, with the content of GM-CSF in the ELISA kit measurement supernatant.The results are shown in Figure 10.Among Figure 10, normal group is the matched group of not dosing; The dosage of five administration groups of oligomannuronic acid is respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml.Vertical coordinate represents the content of GM-CSF among Figure 10.Compare *, P<0.05 with the blank group.
GM-CSF is that formation CFU-GM is necessary, and it can promote that grain-huge biting is the formation of hemopoietic progenitor cell colony.As can be seen from Figure 10, the oligomannuronic acid of 12.5ug/ml and 50ug/ml can obviously promote the stromal cell secrete GM-CSF, with the blank group significant difference is arranged relatively.And effect is the strongest when 25ug/ml.
Embodiment 6, oligomannuronic acid are secreted the effect of IL-3 to splenocyte
Utilize mtt assay to measure oligomannuronic acid to the effect of splenocyte secretion IL-3
The preparation of IL-3 sample: get Kunming mouse, the preparation splenocyte suspension.RPMI-1640 culture fluid with containing 10%FBS is adjusted to 1 * 10 with cell concentration
7/ ml.Get 24 orifice plates, every hole adds cell suspension 875ul, and adding final concentration is the ConA of 5ug/ml, the medicine that adds again variable concentrations makes final concentration be respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml, blank group adds the culture fluid of equivalent.Culture plate is put 37 ℃, 5%CO
2Cultivated 72 hours in the incubator, collect supernatant ,-20 ℃ of preservations are the IL-3 testing sample.
The detection of IL-3 sample: get Kunming mouse, the conventional medullary cell that separates is adjusted cell concentration to 2 * 10 with the RPMI-1640 culture fluid that contains 10%FBS
6/ ml.Get 96 orifice plates, every hole adds 0.1ml cell suspension and 0.1ml IL-3 testing sample, and every sample is established 4 multiple holes.Culture plate is put 37 ℃, 5%CO
2Cultivated 7 days in the incubator, measure cell proliferation with mtt assay.The results are shown in Figure 11.Among Figure 11, normal group is the matched group of not dosing; The dosage of five administration groups of oligomannuronic acid is respectively 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50ug/ml.Vertical coordinate represents absorbance among Figure 11.Compare *, P<0.05 with the blank group; *, P<0.01.
IL-3 claims again the polyclone colony stimulating factor, can promote the proliferation and differentiation of early stage germinal cell, promote the synthetic SCF of marrow stromal cell and IL-6 etc., and SCF and IL-6 can promote propagation and the change of hematopoietic stem cell, CFU-GM.As can be seen from Figure 11, oligomannuronic acid can obviously improve the ability of splenocyte secretion IL-3, compares with the blank group, and significant difference is arranged.And when 12.5ug/ml, act on the strongest.
To sum up, this experimental result shows that oligomannuronic acid can obviously suppress the minimizing of the murine interleukin of cyclophosphamide due to causing, improves the quantity of mouse bone marrow cells nucleated cell, promotes the propagation of mouse bone marrow.No matter Oligomeric manna sugar aldehyde is vivo medicine-feeding or treated in vitro, and normal mouse and cyclophosphamide are suppressed the formation of the CFU-GM of mice, and facilitation is all arranged.Oligomannuronic acid can promote the marrow stromal cell secrete GM-CSF, promotes splenocyte secretion IL-3.Above presentation of results, oligomannuronic acid is many-sided to the effect of hemopoietic system, and the effect of direct promotion proliferation of hematopoietic progenitors is namely arranged, and the indirectly effect by promoting that hematopoieticmicroenviron-ment improves the body hemopoietic function is arranged again.
Because the pharmaceutical salts of oligomannuronic acid has the backbone structure identical with oligomannuronic acid, so its pharmaceutical salts has the application in preparation control leukopenia disease drug identical with oligomannuronic acid.
Rule of origin of the present invention is in Sargassum, have the plurality of advantages such as aboundresources, with low cost and safety be high, prove that in animal level and cellular level it has the effect of better leukocyte increasing, a kind of effective clinically for developing, cheap, the drug provision that has no side effect theoretical foundation.
Above embodiment only is used for technical scheme of the present invention is described, but not limits it; Although with reference to previous embodiment the present invention is had been described in detail, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.